Polymers for delivering peptides and small molecules in vivo

ABSTRACT

Certain hydrophilic polymers, such as a polyoxazoline, when conjugated to a polypeptide or small molecule agent, can enhance the bioavailability of the agent when administered in vivo. Accordingly, hydrophilic polymers of the invention can be used as a delivery vehicle to treat any number of disorders and/or confer a myriad of therapeutic benefits to a subject.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to methods of delivering one or more polypeptideor small molecule therapeutic agents to a cell in conjunction withhydrophilic polymers or hydrophilic polymers and targeting ligands.

2. Background

Many barriers to effective polypeptide and small molecule administrationto in vivo systems have been overcome. For instance, the use of vectors(e.g., viral vectors, polymers, nanoparticles, and liposomes) have showngreat promise in counteracting the rapid rate of clearance of anadministered agent from the blood.

Such delivery systems continue to face obstacles, however. For instance,immunogenicity and toxicity of viral vectors are among the barriers thatlimit their therapeutic application. To this end, a patient's immunedefense often mounts a response to any administered viral vectorparticle, since the viral particles are produced via a natural packagingcell production. Such “natural packaging” produces particles virtuallyidentical to those of the virus from which the vector is derived. Theproduced capsid or envelop, thus, is sensitive and susceptible to hostimmune defenses, which can affectively block the delivery of therecombinant genome.

Previous attempts to overcome these hurdles have involvedtissue-specific targeting of a peptide via a non-invasive or minimallyinvasive route of administration. This approach aids in retainingtherapeutic levels of an agent at the targeted tissue for prolonged timeperiods. Another focus is to allow protein clearance with minimaltoxicity once the desired time period has elapsed.

Administered proteins and polypeptides often suffer from poorbio-availability, due to rapid removal of these molecules from bloodcirculation by enzymatic degradation. One technique for increasingefficacy of protein and other small molecule agents entails conjugatingthe administered agent with a polymer that can provide protection fromenzymatic degradation in vivo, such as a polyethylene glycol (“PEG”)molecule. Such “PEGylation” often improves the circulation time and,hence, bio-availability of an administered agent.

PEG has shortcomings in certain respects, however. For example, becausePEG is a linear polymer, the steric protection afforded by PEG islimited, as compared to branched polymers. Another major shortcoming ofPEG is that it is only amenable to derivatization at its two terminals.This limits the number of other functional molecules (e.g., thosehelpful for protein or drug delivery to specific tissues) that can beconjugated to a PEG.

There is, accordingly, a need for a hydrophilic polymer that is capableof providing superior bioavailability of administered polypeptide- andsmall molecule agents. There also is a need for a hydrophilic polymerthat is compatible with in vivo delivery systems, e.g., vectors, whilemaintaining the foregoing desired properties. The present inventionsatisfies these and other needs.

In addition there is a need to deliver therapeutic agents to specifictissues to achieve maximum therapeutic efficacy while minimizing toxicside effects. The present invention describes the delivery oftherapeutic molecules to specific tissues by ligand-mediated deliverywhere the ligand and the therapeutic molecule are chemically conjugatedto the hydrophilic polymers such as polyoxazoline, polyethylene glycol,polyacetal and others.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide a hydrophilicpolymer that increases bioavailability of a polypeptide or smallmolecule that is administered in vivo.

In another embodiment, the invention provides hydrophilic polymers whichincrease the bioavailibility of agents in an in vivo system, such aspeptides, polypeptides, proteins and small molecules, drugs and nucleicacid drugs.

In another embodiment, the invention provides pharmaceuticalcompositions comprising one or more of the hydrophilic polymersdescribed herein.

In another embodiment, the invention provides methods of increasing thebioavailability of a peptide or small molecule that is administered toan in vivo system, using a hydrophilic polymer.

In another preferred embodiment, the invention also provides methods fordelivering a therapeutic agent to a subject in need thereof comprisingadministering to a subject in need thereof an effective amount of acompound according to any one of claims 1-4.

In another preferred embodiment, the instant polymer comprises atargeting ligand or moiety for targeting specific cells and tissues.

In another preferred embodiment, the instant polymer comprises afusogenic ligand or moiety for facilitating entry of an agent,preferably a nucleic acid, into a nucleus of a cell.

In another preferred embodiment, the instant polymer comprises a nucleartargeting ligand or moiety for targeting specific cells and tissues.

These and other objects will become apparent to a skilled worker byreference to the specification and conventional teachings in the art.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventors surprisingly have discovered that in vivobioavailability of a therapeutic agent can be increased if conjugated toone of a group of hydrophilic polymers. In this sense, the polymer,which preferably is a polyoxazoline, acts as a delivery vehicle for atherapeutic agent. A therapeutic agent is a nucleic acid fragment,peptide, polypeptide, protein or other small molecule drug. By “Peptide”or “polypeptide” is meant two or more amino acids linked to each othervia a peptide bond. As used herein, a “small molecule” or “smallmolecule drug” or “small molecule agent”, or “therapeutic drug” or a“drug” means an organic molecule, other than a nucleic acid moleculethat, when administered to a mammal (e.g., human being), confers atherapeutic benefit. A polymer for use in the invention preferably isconjugated to (i) a nucleic acid, polypeptide or small molecule drug;and (ii) one or more other moieties, e.g., a ligand or tissue-targetingdomain, yet retains (or substantially retains) its desiredcharacteristics. These features, therefore, render the polymersdisclosed herein, e.g., polyoxazolines, polyethylene glycol suitable formultiple routes of administration, ranging from oral, to systemic, tolocal administrations.

As used herein (unless specified to the contrary), the term “polymer” or“polymer of the invention” preferably means a hydrophilic polymerrepresented by any of the following structures.

In a preferred embodiment, a polymer of the invention may be representedby the following:R¹—X—R²

-   -   wherein,    -   X may be a hydrophilic polymer such as polyoxazoline,        polyethylene glycol, polyacetal, polylactic acid, polyglycolic        acid;    -   R¹ may be a hydroxyl group, a sulfhydryl group, carboxylic acid,        carboxylic acid ester, amino, amide group, a cell targeting        ligand, or a tissue targeting ligand; and    -   R² may be a therapeutic agent selected from the group consisting        of a peptide, polypeptide, protein, nucleic acid or a        therapeutic drug,

In another preferred embodiment, a polymer of the invention may berepresented by the following:R²—X—R¹

-   -   wherein,    -   X may be a hydrophilic polymer such as polyoxazoline,        polyethylene glycol, polyacetal, polylactic acid, polyglycolic        acid,    -   R¹ may be a hydroxyl group, a sulfhydryl group, carboxylic acid,        carboxylic acid ester, amino, amide group, a cell targeting        ligand, or a tissue targeting ligand; and    -   R² may be a therapeutic agent selected from the group consisting        of a peptide, polypeptide, protein, nucleic acid or a        therapeutic drug.

In another preferred embodiment, a polymer of the invention may berepresented by the following:

-   -   wherein,    -   X may be —CO—R, —(CH₂)_(m)—COOH, wherein m is an integer 1-25,        (CH₂)_(p)—OH, wherein p is an integer 1-25, —(CH₂)_(q)—COOH,        wherein q is an integer 1-25, an ester group, such as carboxylic        acid esters, polyethylene glycol, polylactic acid, polyglycolic        acid, polyoxazoline, amino, imidazole, or guanidinium; wherein R        may be —CH₃, —C₂H₅, —(CH₂)_(r)—OH, wherein r is an integer 1-25;    -   R may be a hydroxyl group, a sulfhydryl group, carboxylic acid,        carboxylic acid ester, amino, amide group, a targeting moiety, a        fusogenic moiety, or a nuclear targeting moiety; and    -   R² may be a therapeutic agent selected from the group consisting        of a peptide, polypeptide, protein, nucleic acid or a        therapeutic drug, and    -   n is 1-500.

In another preferred embodiment, a polymer of the instant invention mayalso be represented by the following:

-   -   wherein,    -   X may be —CO—R, —(CH₂)_(m)—COOH, wherein m is an integer 1-25,        (CH₂)_(p)—OH, wherein p is an integer 1-25, —(CH₂)_(q)—COOH,        wherein q is an integer 1-25, an ester group, such as carboxylic        acid esters, polyethylene glycol, polylactic acid, polyglycolic        acid, polyoxazoline, amino, imidazole, or guanidinium; wherein R        may be —CH₃, —C₂H₅, or —(CH₂)_(n)—OH, wherein r is an integer        1-25;    -   R¹ may be a therapeutic agent selected from the group consisting        of a peptide, polypeptide, protein, nucleic acid or a        therapeutic drug; and    -   R² may be a hydroxyl group, a sulfhydryl group, carboxylic acid,        carboxylic acid ester, amino, an amide group, a targeting        moiety, a fusogenic moiety, or a nuclear targeting moiety; and    -   n=1-500.

In another preferred embodiment, a polymer of the instant invention mayalso be represented by the following:

-   -   wherein,    -   R may be —CH₃, —C₂H₅, a hydrocarbon with 1-18 carbons,        (C₁-C₂₅)—OH, (C₁-C₂₅)—COOH, polyethyleneglycol, polylactic acid,        polyglycolic acid, polyoxazoline;    -   R¹ may be a hydroxyl group, a sulfhydryl group, carboxylic acid,        carboxylic acid ester, amino, amide group, a cell targeting        ligand, or a tissue targeting ligand; and    -   R² may be a therapeutic agent selected from the group consisting        of a peptide, polypeptide, protein, nucleic acid or a        therapeutic drug, and    -   n=1-500.

In another preferred embodiment, a polymer of the instant invention mayalso be represented by the following:

-   -   wherein,    -   R may be —CH₃, —C₂H₅, a hydrocarbon with 1-18 carbons,        (C₁-C₂₅)—OH, (C₁-C₂₅)—COOH, polyethyleneglycol, polylactic acid,        polyglycolic acid, polyoxazoline;    -   R¹ may be a therapeutic agent selected from the group consisting        of a peptide, polypeptide, protein, nucleic acid or a        therapeutic drug;    -   R² may be a hydroxyl group, a sulfhydryl group, carboxylic acid,        carboxylic acid ester, amino, amide group, a targeting moiety, a        fusogenic moiety or a nuclear targeting moiety, and    -   n=1-500.

In a preferred embodiment, the polymer may be a polyoxazoline, which isa species embraced by the foregoing polymers. A polyoxazoline may berepresented by the following:

-   -   wherein,    -   R may be —CH₃ for polymethyloxazoline (PMOZ), or    -   R may be —CH₂CH₃ for polyethyloxazoline (PEOZ), and    -   n is 1-500.

In the above structure, R¹ is preferably bound to the left side of themolecule.

In another preferred embodiment, a polymer of the instant invention mayalso be represented by the following

-   -   wherein,    -   R¹ may be a therapeutic agent selected from the group consisting        of a peptide, polypeptide, protein, nucleic acid or a        therapeutic drug;    -   R² may be a hydroxyl group, a sulfhydryl group, carboxylic acid,        carboxylic acid ester, amino, amide group, a targeting moiety, a        fusogenic moiety or a nuclear targeting moiety, and    -   n=1-500.

In another preferred embodiment, a polymer of the instant invention mayalso be represented by the following:

-   -   wherein,    -   R¹ may be a hydroxyl group, a sulfhydryl group, carboxylic acid,        carboxylic acid ester, amino, amide group, a cell targeting        ligand, or a tissue targeting ligand; and    -   R² may be a therapeutic agent selected from the group consisting        of a peptide, polypeptide, protein, nucleic acid or a        therapeutic drug, and    -   n=1-500.

Nucleic acid refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides,and peptide-nucleic acids (PNAs).

A polyoxazoline or hydrophilic polymer of the invention also is capableof having multiple ligands conjugated onto the distal ends of thepolymer. This can, for instance, enhance selective tissue and cellularinteractions, thereby minimizing the interaction of an administeredagent and non-targeted tissues and cells.

A targeting ligand enhances binding of the polymer to target tissue orcells and permits highly specific interaction of the polymers with thetarget tissue or cell. In one embodiment, the polymer will include aligand effective for ligand-specific binding to a receptor molecule on atarget tissue and cell surface (Woodle et al., Small molecule ligandsfor targeting long circulating liposomes, in Long Circulating Liposomes:Old drugs, new Therapeutics, Woodle and Storm eds., Springer, 1998, p287-295).

The polymer may include two or more targeting moieties, depending on thecell type that is to be targeted. Use of multiple targeting moieties canprovide additional selectivity in cell targeting, and also cancontribute to higher affinity and/or avidity of binding of the polymerto the target cell. When more than one targeting moiety is present onthe polymer, the relative molar ratio of the targeting moieties may bevaried to provide optimal targeting efficiency. Methods for optimizingcell binding and selectivity in this fashion are known in the art. Theskilled artisan also will recognize that assays for measuring cellselectivity and affinity and efficiency of binding are known in the artand can be used to optimize the nature and quantity of the targetingligand(s).

Suitable ligands include, but are not limited to: vascular endothelialcell growth factor for targeting endothelial cells: FGF2 for targetingvascular lesions and tumors; somatostatin peptides for targeting tumors;transferrin for targeting umors; melanotropin (alpha MSH) peptides fortumor targeting; ApoE and peptides for LDL receptor targeting; vonWillebrand's Factor and peptides for targeting exposed collagen;Adenoviral fiber protein and peptides for targeting Coxsackie-adenoviralreceptor (CAR) expressing cells; PD1 and peptides for targetingNeuropilin 1; EGF and peptides for targeting EGF receptor expressingcells; and RGD containing peptides and their analogues for targetingintegrin expressing cells.

Other examples include (i) folate, where the polymer is intended fortreating tumor cells having cell-surface folate receptors, (ii)pyridoxyl, where the polymer is intended for treating virus-infectedCD4+ lymphocytes, or (iii) sialyl-Lewis, where the polymer is intendedfor treating a region of inflammation. Other peptide ligands may beidentified using methods such as phage display (F. Bartoli et al.,Isolation of peptide ligands for tissue-specific cell surface receptors,in Vector Targeting Strategies for Therapeutic Gene Delivery (Abstractsform Cold Spring Harbor Laboratory 1999 meeting), 1999, p4) andmicrobial display (Georgiou et al., Ultra High Affinity Antibodies fromLibraries Displayed on the Surface of Microorganisms and Screened byFACS, in Vector Targeting Strategies for Therapeutic Gene Delivery(Abstracts form Cold Spring Harbor Laboratory 1999 meeting), 1999, p3.).

In an exemplary embodiment, the targeting ligand may be somatostatin ora somatostatin analog. Somatostatin has the sequence AGCLNFFWKTFTSC, andcontains a disulfide bridge between the cysteine residues. Manysomatostatin analogs that bind to the somatostatin receptor are known inthe art and are suitable for use in the present invention, such as thosedescribed, for example, in U.S. Pat. No. 5,776,894, which isincorporated herein by reference in its entirety. Particularsomatostatin analogs that are useful in the present invention areanalogs having the general structure F*CY-(DW)KTCT, where DW isD-tryptophan and F* indicates, that the phenylalanine residue may haveeither the D- or L-absolute configuration. As in somatostatin itself,these compounds are cyclic due to a disulfide bond between the cysteineresidues. Advantageously, these analogs may be derivatized at the freeamino group of the phenylalanine residue, for example with apolycationic moiety such as a chain of lysine residues. The skilledartisan will recognize that other somatostatin analogs that are known inthe art may advantageously be used in the invention.

Furthermore, methods have been developed to create novel peptidesequences that elicit strong and selective binding for target tissuesand cells such as “DNA Shuffling” (W. P. C. Stremmer, Directed Evolutionof Enzymes and Pathways by DNA Shuffling, in Vector Targeting Strategiesfor Therapeutic Gene Delivery (Abstracts form Cold Spring HarborLaboratory 1999 meeting), 1999, p. 5.) and these novel sequence peptidesare suitable ligands for the invention. Other chemical forms for ligandsare suitable for the invention such as natural carbohydrates which existin numerous forms and are a commonly-used ligand by cells (Kraling etal., Am. J. Path. 150:1307 (1997) as well as novel chemical species,some of which may be analogues of natural ligands such as D-amino acidsand peptidomimetics and others which are identifed through medicinalchemistry techniques such as combinatorial chemistry (P. D. Kassner etal., Ligand Identification via Expression (LIVE): Direct selection ofTargeting Ligands from Combinatorial Libraries, in Vector TargetingStrategies for Therapeutic Gene Delivery (Abstracts form Cold SpringHarbor Laboratory 1999 meeting), 1999, p8.).

The targeting moiety provides tissue- and cell-specific binding. Theligands may be covalently attached to the polymer so that exposure isadequate for tissue and cell binding. For example, a peptide ligand canbe covalently coupled to a polymer such as polyoxazoline or polyethyleneglycol or other hydrophilic polymers.

The number of targeting molecules present on the outer layer will vary,depending on factors such as the avidity of the ligand-receptorinteraction, the relative abundance of the receptor on the target tissueand cell surface, and the relative abundance of the target tissue andcell. Nevertheless, a targeting molecule coupled with each polymerusually provides suitable enhancement of cell targeting.

The presence of the targeting moiety leads to the desired enhancement ofbinding to target tissue and cells. An appropriate assay for suchbinding may be ELISA plate assays, cell culture expression assays, orany other binding assays known in the art.

The fusogenic moiety promotes fusion of the polymer to the cell membraneof the target cell, facilitating entry of the polymer and therapeuticagents into the cell. In one embodiment, the fusogenic moiety comprisesa fusion-promoting element. Such elements interact with cell membranesor endosome membranes in a manner that allows transmembrane movement oflarge molecules or particles, or disrupts the membranes such that theaqueous phases that are separated by the membranes may freely mix.Examples of suitable fusogenic moieties include, but are not limited tomembrane surfactant peptides, e.g. viral fusion proteins such ashemagglutinin (HA) of influenza virus, or peptides derived from toxinssuch as PE and ricin. Other examples include sequences that permitcellular trafficking such as HIV TAT protein and antennapedia or thosederived from numerous other species, or synthetic polymers that exhibitpH sensitive properties such as poly(ethylacrylic acid) (Lackey et al.,Proc. Int. Symp. Control. Rel. Bioact. Mater. 1999, 26, #6245),N-isopropylacrylamide methacrylic acid copolymers (Meyer et al., FEBSLett. 421:61 (1999)), or poly(amidoamine)s, (Richardson et al., Proc.Int. Symp. Control. Rel. Bioact. Mater. 1999, 26, #251), and lipidicagents that are released into the aqueous phase upon binding to thetarget cell or endosome. Suitable membrane surfactant peptides includean influenza hemagglutinin or a viral fusogenic peptide such as theMoloney murine leukemia virus (“MoMuLV” or MLV) envelope (env) proteinor vesicular stroma virus (VSV) G-protein. The membrane-proximalcytoplasmic domain of the MoMuLV env protein may be used. This domain isconserved among a variety of viruses and contains a membrane-inducedα-helix.

Suitable viral fusogenic peptides for the instant invention may includea fusion peptide from a viral envelope protein ectodoinain, amembrane-destabilizing peptide of a viral envelope proteinmembrane-proximal domain, hydrophobic domain peptide segments of socalled viral “fusion” proteins, and an amphiphilic-region containingpeptide. Suitable amphiphilic-region containing peptides include, butare not limited to: melittin, the magainins, fusion segments from H.influenza hemagglutinin (HA) protein, HIV segment I from the cytoplasmictail of HIV1 gp41, and amphiphilic segments from viral env membraneproteins including those from avian leukosis virus (ALV), bovineleukemia virus (BLV), equine infectious anemia (EIA), felineimmunodeficiency virus (FIV), hepatitis virus, herpes simplex virus(HSV) glycoprotein H; human respiratory syncytia virus (hRSV),Mason-Pfizer monkey virus (MPMV), Rous sarcoma virus (RSV),parainfluenza virus (PINF), spleen necrosis virus (SNV), and vesicularstomatitis virus (VSV). Other suitable peptides include microbial andreptilian cytotoxic peptides. The specific peptides or other moleculeshaving greatest utility can be identified using four kinds of assays: 1)ability to disrupt and induce leakage of aqueous markers from liposomescomposed of cell membrane lipids or fragments of cell membranes, 2)ability to induce fusion of liposomes composed of cell membrane lipidsor fragments of cell membranes, 3) ability to induce cytoplasmic releaseof particles added to cells in tissue culture, and 4) ability to enhanceplasmid expression by particles in vivo tissues when administeredlocally or systemically.

The fusogenic moiety also may be comprised of a polymer, includingpeptides and synthetic polymers. In one embodiment, the peptide polymercomprises synthetic peptides containing amphipathic aminoacid sequencessuch as the “GALA” and “KALA” peptides (Wyman T B, Nicol F, Zelphati O,Scoria P V, Plank C, Szoka F C Jr, Biochemistry 1997, 36:3008-3017;Subbarao N K, Parente R A, Szoka F C Jr, Nadasdi L, Pongracz K,Biochemistry 1987 26:2964-2972 or Wyman supra, Subbarao supra). Otherpeptides include non-natural aminoacids, including D aminoacids andchemical analogues such as peptoids. Suitable polymers include moleculescontaining amino or imidazole moieties with intermittent carboxylic acidfunctionalities such as ones that form “salt-bridges,” either internallyor externally, including forms where the bridging is pH sensitive. Otherpolymers can be used including ones having disulfide bridges eitherinternally or between polymers such that the disulfide bridges blockfusogenicity and then bridges are cleaved within the tissue orintracellular compartment so that the fusogenic properties are expressedat those desired sites. For example, a polymer that forms weakelectrostatic interactions with a positively charged fusogenic polymerthat neutralizes the positive charge could be held in place withdisulfide bridges between the two molecules and these disulfides cleavedwithin an endosome so that the two molecules dissociate releasing thepositive charge and fusogenic activity. Another form of this type offusogenic agent has the two properties localized onto different segmentsof the same molecule and thus the bridge is intramolecular so that itsdissociation results in a structural change in the molecule. Yet anotherform of this type of fusogenic agent has a pH sensitive bridge.

The fusogenic moiety also may comprise a membrane surfactantpolymer-lipid conjugate. Suitable conjugates include Thesit™, Brij 58™,Brij 78™, Tween 80™, Tween 20™, C₁₂E₈, C₁₄E₈, C₁₆E₈(C_(n)E_(n),=hydrocarbon poly(ethylene glycol)ether where C representshydrocarbon of carbon length N and E represents poly(ethylene glycol) ofdegree of polymerization N), Chol-PEG 900, analogues containingpolyoxazoline or other hydrophilic polymers substituted for the PEG, andanalogues having fluorocarbons substituted for the hydrocarbon.Advantageously, the polymer will be either biodegradable or ofsufficiently small molecular weight that it can be excreted withoutmetabolism. The skilled artisan will recognize that other fusogenicmoieties also may be used without departing from the spirit of theinvention.

Certain therapeutic agents exert their biological activity in the cellnucleus. Advantageously, when the intended biological target of anucleic acid is the nucleus, the nucleic targeting moiety of theinvention is “nuclear targeted,” that is, it contains one or moremolecules that facilitate entry of the nucleic acid through the nuclearmembrane into the nucleus of the host cell. Such nuclear targeting maybe achieved by incorporating a nuclear membrane transport peptide, ornuclear localization signal (“NLS”) peptide, or small molecule thatprovides the same NLS function, into the core complex. Suitable peptidesare described in, for example, U.S. Pat. Nos. 5,795,587 and 5,670,347and in patent application WO 9858955, which are hereby incorporated byreference in their entirety, and in Aronsohn et al., J. Drug Targeting1:163 (1997); Zanta et al., Proc. Nat'l Acad. Sci. USA 96:91-96 (1999);Ciolina et al., Targeting of Plasmid DNA to Importin alpha by Chemicalcoupling with Nuclear Localization SigIal Peptides, in Vector TargetingStrategies for Therapeutic Gene Delivery (Abstracts from Cold SpringHarbor Laboratory 1999 meeting), 1999, p 20; Saphire et al., J. BiolChem; 273:29764 (1999). A nuclear targeting peptide may be a nuclearlocalization signal peptide or nuclear membrane transport peptide and itmay be comprised of natural aminoacids or non-natural ammoacidsincluding D aminoacids and chemical analogues such as peptoids. The NLSmay be comprised of aminoacids or their analogues in a natural sequenceor in reverse sequence. Another embodiment provides a steroidreceptor-binding NLS moiety that activates nuclear transport of thereceptor from the cytoplasm, wherein this transport carries the nucleicacid with the receptor into the nucleus.

In another embodiment, the NLS is coupled to the polymer in such amanner that the polymer is directed to the cell nucleus where it permitsentry of a nucleic acid into the nucleus.

In another embodiment, incorporation of the NLS moiety into the polymeroccurs through association with the nucleic acid, and this associationis retained within the cytoplasm. This minimizes loss of the NLSfunction due to dissociation with the nucleic acid and ensures that ahigh level of the nucleic acid is delivered to the nucleus. Furthermore,the association with the nucleic acid does not inhibit the intendedbiological activity within the nucleus once the nucleic acid isdelivered.

In yet another embodiment, the intended target of the biologicalactivity of the nucleic acid is the cytoplasm or an organelle in thecytoplasm such as ribosomes, the golgi apparatus, or the endoplasmicreticulum. In this embodiment, a localization signal is included in thepolymer or anchored to it so that it provides direction of the nucleicacid to the intended site where the nucleic acid exerts its activity.Signal peptides that can achieve such targeting are known in the art.

By virtue of its structure and chemical properties, a polymer of theinvention, for example polyoxazoline, can provide advantages overconventionally used hydrophilic polymers, such as PEG. Preferredpolyoxazolines of the inventions are polymethyloxazoline andpolyethyloxazoline.

A polymer of the invention can be constructed and used as a linearpolymer. Such polymers provide several advantages over commonly usedhydrophilic polymer, such a PEG. For instance, polyoxazoline, whenattached to a therapeutic agent, can provide longer blood circulationtime for the agent. A polyoxazoline or other polymer of the inventionalso can provide hydrophilicity to a hydrophobic drug, which enhancesbioavailability of the drug. The nitrogens in the back bone are amenableto substitutions. A PEG polymer backbone, on the other hand, containsoxygen atoms that are as amenable to substitutions. Accordingly, thehydrophobicity of a polyoxazoline can be modulated according toconventional means. Atoms such as nitrogens in the backbone also can beused to attach other functional molecules such as ligands, which cantarget the therapeutic agent or drug to specific tissues and cells.

Nitrogens in the backbone also can be used to introduce branching of thepolymer. It is expected that the network of branching will protect anadministered agent to a greater extent in vivo than other polymers(e.g., PEG), which will result in enhanced bioavailability. The branchedstructure of a polyoxazoline or other polymer of the invention, thus,can provide a desired effect on pharmacology, such as: increasedcirculation in the blood, increased affinity for cellular binding anduptake, and/or facilitated adsorption into the tissue and cell.

As indicated, functional moieties can be added to a polymer, e.g.,polyoxazoline, at the nitrogens in the polymer backbone, the latterbeing amenable to derivatization. Functional moieties suitable forattachment to a polyoxazoline (either alone or in comibination) include:vascular endothelial growth factors, somatostatin and somatostatinanalogs, transferring, melanotropin, ApoE and ApoE peptides, vonWillebrand's factor and von Willebrand's factor peptides, adeno viralfiber protein and adenoviral fiber protein peptides, PD1 and PD1peptides, EGF and EGF peptides, RGD peptides, CCK peptides, antibody andantibody fragments, folate, pyridoxyl and sialyl-Lewis X and chemicalanalogs thereof. A polypeptide or other small molecule preferably isattached at the terminal ends of the polymer.

Polymers of the present invention can possess one or more asymmetriccarbon atoms and are thus capable of existing in the form of opticalisomers as well as in the form of racemic or nonracemic mixturesthereof. The optical isomers can be obtained by resolution of theracemic mixtures according to conventional processes, for example byformation of diastereoisomeric salts by treatment with an opticallyactive acid or base. Examples of appropriate acids are tartaric,diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric andcamphorsulfonic acid and then separation of the mixture ofdiastereoisomers by crystallization followed by liberation of theoptically active bases from these salts. A different process forseparation of optical isomers involves the use of a chiralchromatography column optimally chosen to maximize the separation of theenantiomers. Still another available method involves synthesis ofcovalent diastereoisomeric molecules by reacting the instant polymerswith an optically pure acid in an activated form or an optically pureisocyanate. The synthesized diastereoisomers can be separated byconventional means such as chromatography, distillation, crystallizationor sublimation, and then hydrolyzed to deliver the enantiomerically purecompound. The optically active compounds can likewise be obtained byutilizing optically active starting materials. These isomers may be inthe form of a free acid, a free base, an ester or a salt.

The compounds of the present invention can be used in the form of saltsderived from inorganic or organic acids. These salts include but are notlimited to the following: acetate, adipate, alginate, citrate,aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate,camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate,ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate,heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxy-ethanesulfonate, lactate, maleate,methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate, mesylate andundecanoate. Also, the basic nitrogen-containing groups can bequaternized with such agents as lower alkyl halides, such as methyl,ethyl, propyl, and butyl chloride, bromides, and iodides; dialkylsulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides, aralkyl halides like benzyl and phenethylbromides, and other. Water or oil-soluble or dispersible products arethereby obtained.

Examples of acids which may be employed to form pharmaceuticallyacceptable acid addition salts include such inorganic acids ashydrochloric acid, sulphuric acid and phosphoric acid and such organicacids as oxalic acid, maleic acid, succinic acid and citric acid. Otherexamples include salts with alkali metals or alkaline earth metals, suchas sodium, potassium, calcium or magnesium or with organic bases.

Methods of Making Polyoxazoline-Agent Conjugates.

A polymer of the invention, such as polyoxazoline, is conjugated, e.g.,chemically, to a polypeptide or small molecule agent prior to in vivoadministration. With reference to the following structures, thefollowing protocol is a non-limiting method of preparing apolyoxazoline-agent conjugate of the invention.

Polymerization of the 2-oxazoline can be carried out using ICH₂CO₂Et asthe initiator. The monomer (0.1 mmol) is taken in a dry glass tube andan equal volume of dry acetonitrile is added thereto. Depending on thedegree of polymerization desired, a suitable amount of initiator isadded to the above solution (e.g., 0.001 for a degree of polymerizationof 100). The mixture is sealed under nitrogen at 80° for about 24 hours.Since oxazoline polymerization does not terminate without a chainterminator, a methanolic solution of KOH (0.5M) is added as chainterminator.

The resulting polyoxazoline with a carboxylic acid end can be conjugatedto a free amino group or a hydroxyl group of a peptide, protein or drugas follows. To a 1 molar equivalent of the polymer (based on thecarboxylic acid residue) is added 1.1 molar equivalents of dicyclohexylcarbodiimide(DCCI) and N-hydroxysuccinimide (NHS) at 0° C. To this, 1molar equivalent of peptide or protein is added. The mixture is stirredat 0° C. for about one hour and then overnight at room temperature. Thewhite precipitate of DCHU, is filtered off and the filtrate isevaporated to dryness. The conjugate is redissolved in water anddialyzed to remove small molecule impurities.

A polyoxazoline also can be conjugated to one or more moieties, such astissue targeting molecules, including: vascular endothelial growthfactors, somatostatin and somatostatin analogs, transferrin,melanotropin, ApoE and ApoE peptides, von Willebrand's factor and vonWillebrand's factor peptides, adeno viral fiber protein and adenoviralfiber protein peptides, PD1 and PD1 peptides, EGF and EGF peptides, RGDpeptides, CCK peptides, antibody and antibody fragments, folate,pyridoxyl and sialyl-Lewis X and chemical analogs thereof. Polyoxazolinecan also be conjugated to endosome disrupting molecules such asfusogenic moiety of a viral peptide selected from the group consistingof MLV envelope protein, HA env peptide, a viral envelope proteinectodomain, a membrane—destabilizing domain of viral envelope protein,and hydrophobic domain of a viral fusion protein.

These molecules can be attached to the end of the polymer that isopposite to the end where the therapeutic molecule is attached. Theorder of attachment and synthetic strategies, which will be apparent toskilled worker in, the field, is determined based on the chemicalproperties of the molecules to be attached. To attach peptides andproteins, synthetic schemes similar to those shown in the abovedescribed protocol may be employed. For other molecules, the skilledartisan will recognize that other synthetic strategies will be useddepending on the functional groups available for conjugation on thesemolecules.

Therapeutic Methods.

A polymer-agent conjugate, such as an agent-polyoxazoline conjugate(optionally attached to other moieties), can be used in a variety waysto bring about a therapeutic effect. A polyoxazoline-agent conjugate isparticularly suitable for delivering an effective amount of atherapeutic agent to an in vivo system over an extended period of time.This finding is significant, given the limitations of state of the artdelivery compositions. As a result, the drug and gene delivery vehiclesof the invention can be useful in a number of therapeutic applications,including: therapeutic vaccines, preventative vaccines, treatment ofinflammatory disorders and many types of malignancies, as well as anyother regimen involving repeated administration of a therapeutic agent,which is any agent which elicits a beneficial response or alleviatessymptoms of a disease or disorder and includes peptides, polypeptides,proteins, nucleic acids and small molecule drugs.

The present invention provides methods of administering one or moretherapeutic small molecules or polypeptides to a subject, using avehicle comprised of a polyoxazoline, to bring about a therapeuticbenefit to the subject. As used herein, a “therapeutic small molecule”or “therapeutic polypeptide” is any small molecule or polypeptide thatcan confer a therapeutic benefit to a subject. In the present invention,a therapeutic small molecule or polypeptide also can be administered toa subject in conjunction with a synthetic vector. The subject preferablyis mammalian such as a mouse, and more preferably is a human being.

Delivery vehicles for use in the present invention can be used tostimulate an immune response, which may be protective or therapeutic.Accordingly, the delivery vehicles can be used to vaccinate a subjectagainst an antigen.

In this sense, the invention provides methods for vaccinating orenhancing a physiological response against a pathogen in a subject. Thismethodology can entail administering to the subject a first, or priming,dosage of a therapeutic peptide, followed by administering to thesubject one or more booster dosages of the therapeutic peptide.

The administration regimen can vary, depending on, for example, (i) thesubject to whom the therapeutic agent is administered, and (ii) thepathogen that is involved. For instance, a booster dosage of atherapeutic peptide may administered about two weeks after priming,followed by successive booster dosages, which can occur betweenintervals of constant or increasing duration. It is desirable toadminister therapeutic peptide molecules at a periodicity that isappropriate according to the subject's immune response.

In the preceding administration steps, the administered peptide moleculeis conjugated to a polymer of the invention. Preferably, the therapeuticpeptide molecule in the foregoing steps elicits a humoral and/orcellular response in the subject, causing the subject to exhibit adegree of immunity against the pathogen that is greater than before thetherapeutic method is carried out.

The antigen against which the subject exhibits an increased immunity canbe the peptide antigen that is administered. Alternatively, the antigenagainst which the subject exhibits an increased immunity is distinctfrom, or in addition to, the administered peptide antigen. In the latterapproach, for instance, the peptide antigen can act to enhance an immuneresponse against another antigen, e.g., a component of a tumor.

The route of administration may vary, depending on the therapeuticapplication (e.g., preventative or therapeutic vaccine) and the type ofdisorder to be treated. The peptide delivery vehicle may be injectedinto the skin, muscle, intravenously, directly to the portal, hepaticvein or bile duct, locally to a tumor or to a joint, or orally.

An administered therapeutic peptide molecule also may induce an immuneresponse. A response can be achieved to intracellular infectious agentsincluding, for example, tuberculosis, Lyme disease, and others. Aresponse can be achieved by delivery of an antigen, cytokines, or acombination thereof. The invention also provides for the delivery of HIVantigens and induction of both a protective and a therapeutic immuneresponse for preventing and treating HIV, respectively.

The invention additionally provides for the delivery of antigens thatelicit a humoral and/or a cellular immune response. This heightenedimmune response can provide protection from a challenge with infectiousagents characterized as containing or displaying the antigen. In oneembodiment, the therapeutic agent is a cytokine, which may or may not beco-administered with another antigen. A cytokine acts to recruit animmune response, which can enhance an immune response to an expressedantigen. Accordingly, cytokine administration according to the inventioncan induce APCs and other immune response cells to the vicinity of tumorcells, in which case there is no requirement for co-adminstration of anantigen. Yet, in another embodiment, one or more antigens and cytokinescan be co-administered.

Accordingly, the invention contemplates the use of immunostimulatorycytokines, as well as protein analogues exhibiting biological activitysimilar to an immunostimulatory cytokine, to vaccinate a subject.Suitable cytokines for use in enhancing an immune response includeGM-CSF, IL-1, IL-12, IL-15, IL-2, interferons, B-40, B-7, tumor necrosisfactor (TNF) and others. The invention also contemplates utilizingtherapeutic agents that can down-regulate immunosupressant cytokines.The invention also provides for administration of “recruitmentcytokines” at tumors, which can initiate a cellular immune response atthe tumor site, giving recognition and killing of tumor cells at thesite of expression and at distal tumor sites.

A polyoxazoline also may be used to deliver an agent that treats adisorder characterized by inflammation. In one approach, one or moretherapeutic agents is administered to a subject suffering from adisorder characterized by inflammation, in order to suppress or retardan immune response. Treatable disorders include rheumatoid arthritis,psoriasis, gout and inflammatory bowel disorders. Suitable therapeuticagents for use in treating inflammation include inflammation inhibitorycytokines, such as: IL-IRA, soluble TNF receptor, and soluble Fasligand.

The route and site of administration will vary, depending on thedisorder and the location of inflammation. The polyoxazoline-agent, withor without a synthetic vector, can be administered into a joint;administration thereto can be in conjunction with electroporation.

Pharmaceutical Compositions

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration.

The dosage regimen for treating a disease condition with the compoundsand/or compositions of this invention is selected in accordance with avariety of factors, including the type, age, weight, sex, diet andmedical condition of the patient, the severity of the disease, the routeof administration, pharmacological considerations such as the activity,efficacy, pharmacokinetic and toxicology profiles of the particularcompound employed, whether a drug delivery system is utilized andwhether the compound is administered as part of a drug combination.Thus, the dosage regimen actually employed may vary widely and thereforemay deviate from the preferred dosage regimen set forth above.

The compounds of the present invention may be administered orally,parenterally, by inhalation spray, rectally, or topically in dosage unitformulations containing conventional nontoxic pharmaceuticallyacceptable carriers, adjuvants, and vehicles as desired. Topicaladministration may also involve the use of transdermal administrationsuch as transdermal patches or iontophoresis devices. The termparenteral as used herein includes subcutaneous injections, intravenous,intramuscular, intrasternal injection, or infusion techniques.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

Suppositories for rectal administration of the drug can be prepared bymixing the drug with a suitable nonirritating excipient such as cocoabutter and polyethylene glycols which are solid at ordinary temperaturesbut liquid at the rectal temperature and will therefore melt in therectum and release the drug.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound may be admixed with at least one inert diluent such assucrose lactose or starch. Such dosage forms may also comprise, as innormal practice, additional substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets, and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally by prepared with enteric coatings.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, and sweetening, flavoring, andperfuming agents.

While the compounds of the invention can be administered as the soleactive pharmaceutical agent, they can also be used in combination withone or more therapeutic agents, such as immunomodulators, antiviralagents or antiinfective agents.

The foregoing is merely illustrative of the invention and is notintended to limit the invention to the disclosed compounds. Variationsand changes which are obvious to one skilled in the art are intended tobe within the scope and nature of the invention which are defined in theappended claims. From the foregoing description, one skilled in the artcan easily ascertain the essential characteristics of this invention,and without departing from the spirit and scope thereof, can makevarious changes and modifications of the invention to adapt it tovarious usages and conditions.

All documents referred to herein are specifically incorporated herein byreference in their entireties, including the priority document, U.S.Provisional Application No. 60/352,881, filed Feb. 1, 2002, which isincorporated herein by reference in its entirety.

1. A compound represented by the following structure:R¹ —X—R² wherein, X is a hydrophilic polymer selected from the groupconsisting of polyoxazoline, polyethylene glycol, polyacetal, polylacticacid, and polyglycolic acid; R¹ is a hydroxyl group, a sulfhydryl group,carboxylic acid, carboxylic acid ester, amino, amide group, a celltargeting ligand, or a tissue targeting ligand; and R² may be atherapeutic agent selected from the group consisting of a peptide,polypeptide, protein, nucleic acid and a therapeutic drug,
 2. A compoundrepresented by the following structure:R¹—X—R² wherein, X is a hydrophilic polymer selected from the groupconsisting of polyoxazoline, polyethylene glycol, polyacetal, polylacticacid, polyglycolic acid, R1 is a therapeutic agent selected from thegroup consisting of a peptide, polypeptide, protein, nucleic acid and atherapeutic drug; and R2 is a hydroxyl group, a sulfhydryl group,carboxylic acid, carboxylic acid ester, amino, amide group, a celltargeting ligand, or a tissue targeting ligand.
 3. A compoundrepresented by the following structure:

wherein, R¹ is a therapeutic agent selected from the group consisting ofa peptide, polypeptide, protein, nucleic acid and a therapeutic drug; R²is a hydroxyl group, a sulfhydryl group, carboxylic acid, carboxylicacid ester, amino, amide group, a targeting moiety, a fusogenic moietyor a nuclear targeting moiety, and n=1-500.
 4. A compound represented bythe following structure:

wherein, R¹ is a hydroxyl group, a sulfhydryl group, carboxylic acid,carboxylic acid ester, amino, amide group, a cell targeting ligand, or atissue targeting ligand; R² is a therapeutic agent selected from thegroup consisting of a peptide, polypeptide, protein, nucleic acid and atherapeutic drug, and n=1-500.
 5. A compound represented by thefollowing structure:

wherein, X is —CO—R, —(CH₂)_(m)—COOH, wherein m is an integer 1-25,(CH₂)_(p)—OH, wherein p is an integer 1-25, —(CH₂)_(q)—COOH, wherein qis an integer 1-25, an ester group, polyethylene glycol, polylacticacid, polyglycolic acid, polyoxazoline, amino, imidazole, orguanidinium; wherein R may be —CH₃, —C₂H₅, —(CH₂)_(r)—OH, wherein r isan integer 1-25; R¹ is a hydroxyl group, a sulfhydryl group, carboxylicacid, carboxylic acid ester, amino, amide group, a targeting moiety, afusogenic moiety, or a nuclear targeting moiety; and R² is a therapeuticagent selected from the group consisting of a peptide, polypeptide,protein, nucleic acid or a therapeutic drug, and n is 1-500.
 6. Acompound represented by the following structure:

wherein, X is —CO—R, —(CH₂)_(m)—COOH, wherein m is an integer 1-25,(CH₂)_(p)—OH, wherein p is an integer 1-25, —(CH₂)_(q)—COOH, wherein qis an integer 1-25, an ester group, polyethylene glycol, polylacticacid, polyglycolic acid, polyoxazoline, amino, imidazole, orguanidinium; wherein R may be —CH₃, —C₂H₅, —(CH₂)_(r)—OH, wherein r isan integer 1-25; R¹ is a therapeutic agent selected from the groupconsisting of a peptide, polypeptide, protein, nucleic acid or atherapeutic drug; and R² is a hydroxyl group, a sulfhydryl group,carboxylic acid, carboxylic acid ester, amino, an amide group, atargeting moiety, a fusogenic moiety, or a nuclear targeting moiety; andn=1-500.
 7. A compound represented by the following structure:

wherein, R is —CH₃, —C₂H₅, a hydrocarbon with 1-18 carbons, (C₁-C₂₅)—OH,(C₁-C₂₅)—COOH, polyethyleneglycol, polylactic acid, polyglycolic acid,polyoxazoline; R¹ is a hydroxy group, a sulfhydryl group, carboxylicacid, carboxylic acid ester, amino, amide group, a cell targetingligand, or a tissue targeting ligand; and R² is a therapeutic agentselected from the group consisting of a peptide, polypeptide, protein,nucleic acid or a therapeutic drug, and n=1-500.
 8. A compoundrepresented by the following structure:

wherein, R is —CH₃, —C2H₅, a hydrocarbon with 1-18 carbons, (C₁-C₂₅)—OH,(C₁-C₂₅)—COOH, polyethyleneglycol, polylactic acid, polyglycolic acid,polyoxazoline; R¹ is a therapeutic agent selected from the groupconsisting of a peptide, polypeptide, protein, nucleic acid or atherapeutic drug; R² is a hydroxyl group, a sulfhydryl group, carboxylicacid, carboxylic acid ester, amino, amide group, a targeting moiety, afusogenic moiety or a nuclear targeting moiety, and n=1-500.
 9. A methodof treating a subject suffering from a disorder treatable by atherapeutic agent, comprising administering to said subject an effectiveamount of a compound according to any one of claims 1-8.
 10. A methoddelivering a therapeutic agent to a subject in need thereof comprisingadministering to a subject in need thereof an effective amount of acompound according to any one of claims 1-8.